Our hypothesis is that near-infrared (NIR) fluorescence has the potential to revolutionize image-guided surgery. At the beginning of our first award period, there were neither optimized imaging systems nor optimized contrast agents to test this hypothesis. Nevertheless, in only 31/2 years, our Bioengineering Research Partnership (BRP), comprised of the Frangioni Laboratory at the Beth Israel Deaconess Medical Center (BIDMC), GE Healthcare, and Siemens Corporate Research, has designed, constructed, and disseminated the FLARE (Fluorescence-Assisted Resection and Exploration) intraoperative NIR fluorescence imaging system. By exploiting several innovations in hardware and software, FLARE is capable of visualizing two independent channels of NIR fluorescence in real-time, with high sensitivity and resolution, and simultaneously with surgical anatomy. In addition to validating the technology in over 200 rodent and 100 large animal surgeries, we have exceeded the original Specific Aims by translating FLARE to the clinic where it is now being used in three separate, NIH-funded clinical trials in breast cancer SLN mapping, lung cancer SLN mapping, and perforator vessel mapping. We have also leveraged our resources to add the Patonay/Strekowski Laboratory at Georgia State University (GSU), recognized leaders in NIR fluorophore chemistry, to our BRP and with them have developed ultra-low background heptamethine indocyanines for potential clinical translation. Despite these advances, there remain four fundamental areas in the field that require ongoing investigation: 1) the development of minimally-invasive (e.g., laparoscopy/endoscopy) NIR fluorescence technology for the approximately 50% of human surgeries that are no longer performed using open incisions, 2) the addition of spatially-modulated light capabilities to quantify tissue optical properties, especially tissue oxygenation, in the absence of exogenous NIR fluorophores, 3) the addition of fluorescence lifetime imaging (FLIM) capability to FLARE to improve separation of exogenous NIR fluorophores from tissue autofluorescence, and 4) the first-in-human testing of novel NIR fluorophores that will lay the foundation for future disease-specific contrast agents. We focus the Specific Aims of this competing renewal application on these four key areas, and have updated our BRP to include Qioptiq LINOS, Inc., experts in optical engineering, Yankee Modern Engineering, experts in medical device mechanical engineering, Albright Technologies, Inc., experts in micro-fabrication and silicone heat dissipation technology, the Electronics Design Facility of Boston University, experts in laser control electronics and digital light processing (DLP), and Seres Laboratories, one of the few cGMP chemistry contractors with experience in producing heptamethine indocyanines. GSU will continue to assist with cGMP scale-up. Finally, we propose to intensify clinical translation activities during the second award period, including first-in-human trials of the engineering and chemistry innovations that arise from this BRP.